Cancer cells exhibit a unique metabolic profile characterized by increased glycolysis with lactate generation and a decreased reliance on mitochondrial respiration regardless of oxygen availability. Significantly, recent work in our lab demonstrates that expression of the pyruvate kinase-M2 (PK-M2) isoform is necessary for this metabolic phenotype. Additionally, all tumors evaluated exclusively express PK-M2, indicating that PK- M splice variant expression may be important for tumor development, progression and/or maintenance. Expression of PK-M2 also provides a tumorigenic advantage over cells exclusively expressing PK-M1. The factors that govern alternative splicing of PK-M and the enhanced tumorigenicity of PK-M2-expressing cells are not understood. This proposal seeks to (1) establish a system which can be utilized to investigate alternative splicing of PK-M, (2) identify splice and regulatory factors governing PK-M splice variant expression and (3) investigate the mechanism whereby PK-M2 expression confers a tumorigenic advantage in vivo. Understanding these processes will provide insight into tumor cell metabolism and may have therapeutical implications. The system which will be utilized to investigate PK-M alternative splicing will consist of a differentiation model, where the switch in PK-M isoform expression can be observed, a fluorescent PK-M2 splice reporter, which will permit monitoring of PK-M isoform expression by fluorescence, and a PK-M mini-gene, which will be used for validation of putative regulatory factors. We will then utilize these tools to conduct a loss-of- function candidate screen with a custom arrayed shRNA library containing factors with functionally significant domains and characterized roles in alternative splicing to identify factors governing PK-M isoform expression. Lastly, we will investigate the tumorigenic advantage conferred by PK-M2 expression by conducting a DMA microarray to compare expression profiles of cancer cells exclusively expressing PK-M1 or PK-M2. Investigating the regulation of the mechanism dictating which of two different forms of a specific metabolic enzyme is made will lead to a greater understanding of cancer cell metabolism. This understanding could identify novel therapeutic approaches by which the unique metabolism of cancer cells, in contrast to normal cell counterparts, could be targeted in the clinic.